臺灣白芷組織培養之研究（2）——細胞懸浮培養之建立及培養條件之探討

Studies on the Tissue Culture of Angelica Dahurica Var. Formosana Ⅱ. Establishment of Cell Suspension Culture and Evaluation of Cultural Conditions

找出適當培養條件，建立生長快速的懸浮細胞，是利用懸浮細胞生產二次代謝產物首需解決的問題。本研究利用臺灣白芷藥柄所誘導的癒合組織建立懸浮細胞，並找出此懸浮細胞生長之最佳條件，結果顯示，以含有全量或半量之Murashige and Skoog (1962)基本鹽類添加的0.5~4mg/l 2,4-D、0.5mg/l kinetin及3~5% sucrose，pH=6~7之液體培養基，並於200lux光照，25±1℃恆溫及120rpm轉速下培養，可得一分散均勻，生長良好之懸浮細胞。懸浮培養的初體積影響細胞的生長速率，在25ml的培養基中以5ml PCV（packed cell volume，細胞沉降體積）之初體積生長最快速，約12天後可達生長靜止翔，繼代培養宜早於此時間；但如欲長期繼代培養，則較少之初體積(0.5~1ml PCV)培養，約22天後始達生長靜止期，可減少更換培養液的次數。本試驗建立之白芷懸浮細胞，應可作為生產二次代謝物imperatorin（臺灣白芷之主要藥用成份之一）之原料。

The establishment of a successful cell suspension culture system is a prerequisite for the production of secondary metabolites from medicinal herbs. The purpose of this study was to establish the cell suspension culture from petioles of Angelica dahurica var. formosana and to evaluate the suitable cultural condition for the suspension cell growth. It was found that for developing a rapidly growing and finely dispersed cell suspension culture, the best medium composition was 1/2 MS basal salts, 0.5-4mg/l 2, 4-D, 0.5mg/l kinetin and 3-5% sucrose with medium pH of 6-7. The appropriate light intensity was 200lux with a photoperiod of 16 hr cultured under 25±1℃ condition. The shaking speed was suggested to be 120 rpm. The initial volume affected significantly the growth rate of cells in suspension. The stationary growth phase was reached within 12 days with an initial volume of 5ml packed cells in 25ml medium. However, lower volume (0.5-1.0ml) was suggested for long-term suspension culture as the stationary phase was detained to 22 days after culture. In a typical growth cycle of the suspension cells, the stationary phase was reached around the 16th day, whereas the production of imperatorin, the major medicinal compound produced by the culture, was maximum at the 12th day. The cell suspension of A. dahurica var. formosana established from this study could be used as a pilot model for the production of imperatorin.